The Mechanism Of Colon Epithelial Injury Induced By Chronic Stress And The Effect Of Melatonin On Alleviating Injury

Intestinal mucosa as the first line of defense against the invasion of pathogenic bacteria is critical for animal health.In ruminants,compared to stratified ruminal epithelium,the hindgut as monolayer columnar epithelium is more susceptible to harmful molecules.The negative effects of chronic stress on gut health has attracted more and more attention.Under stress condition,the dysfunction of intestinal ecological balance is one of the important reasons that threaten animal health.The colon is also the site of the greatest accumulation of toxins,which can directly damage lung epithelium.It’s well known that Pseudomonas aeruginosa is common intestinal bacteria,and pyocyanin(PCN)is a major toxin factor,and also acts as a quorum sensing molecule to coordinate bacteria behavior.Melatonin(MEL)is synthesized and secreted by pineal gland,and a large amount of MEL can also be synthesized by intestinal epithelial cells.MEL can protect the intestinal tract from the attack of antigens and toxins through regulating microbial flora and circadian rhythm.Up to now,there are few reports about the impact of chronic stress on intestinal barrier and circadian rhythm in ruminants.Based on the previous report,long-term low dosage of dexamethasone(DEX)treatment was used to mimic the chronic stress response.Therefore,this study was aimed to explore the effect of chronic stress on intestinal cells and barrier status,and to elucidate the mechanism through clock gene expression,MEL secretion,luminal harmful products and oxidative response,and to further reveal whether an enhancement of MEL level could alleviate the injury induced by luminal PCN.Our research will be helpful for understanding the impact of chronic stress on gut health,and providing references for health management in ruminants.The results are as follows:1 Effects of chronic stress on the colonic epithelium structure of goats and its mechanismIn this study,ten six-month-old Boer goats(25.0±1.0 kg bodyweight)were selected and raised in separate corral.Animals were fed daily at 08:00 h and 18:00 h,respectively,and free access to water.Ten goats were randomly divided into two groups,control group(Con,n=5)and DEX treated group(DEX,n=5).DEX(0.2 mg/kg)was daily injected intramuscularly at 08:00 for 21 days,goats in Con group were injected with same volume of saline.At the end of experiment,animals were slaughtered after overnight fasting,then samples including blood,colonic epithelial tissues and colonic digesta were collected for analysis.The results showed that compared to Con,DEX treatment significantly increased pyocyanin(PCN)content in colonic digesta(P<0.05),and activated colonic epithelial apoptosis(P<0.05).The activity of caspase-3 and 9 in colonic epithelium was significantly increased by DEX(P<0.05),and TLR4 and NF-κB-p65 protein expression was also greatly up-regulated(P<0.05).However,DEX significantly down-regulated CCND2 and PCNA expression(P<0.05),as well as the tight junction protein claudin-4 expression(P<0.05).Real-time PCR results showed that apoptosis related genes including Caspase-3 and 8,proliferation related genes of CDK1,CDK2,CDK4,CCND1 and CCND2,tight junction genes of Claudin1,Claudin 4 and ZO-1,and inflammatory genes of IL-1,IL-10,and iNOS were generally up-regulated by DEX in colonic epithelium(P<0.05).We found that the overall DNA methylation of genomic DNA in DEX group was significantly decreased compared to Con(P<0.05),and the methylation level of the target gene promoter was also significantly decreased(P<0.05),which was consistent with the up-regulation of target genes expression in DEX group.The above results showed that chronic stress induced by DEX damaged the integrity of intestinal barrier,changed the level of DNA methylation and gene expression profile in intestinal epithelium.2 Effects of chronic stress on melatonin concentration and intestinal biological clock genes expression in goatsPlasma melatonin concentration was measured by ELISA kit.The results showed that DEX treatment decreased blood melatonin concentration significantly(P<0.05).Compared to Con,DEX significantly decreased the antioxidant enzyme GSH activity in plasma(P<0.05),while MDA content increased significantly(P<0.05).The level of CLOCK,BMAL1,Per1,Per2 and Npas2 genes expression was significantly up-regulated by DEX in the hypothalamus,colonic and cecal epithelial tissues(P<0.05),but not in adrenal gland,pituitary gland,duodenum and ruminal epithelial tissues(P>0.05).Melatonin receptor 2(MT2)and glucocorticoid receptor(GR)expression was significantly up-regulated in colonic and cecal epithelium by DEX(P<0.05).Compared to Con,MT2 protein expression was significantly decreased in hypothalamus(P<0.05),but up-regulated in colonic and cecal epithelium(P<0.05)in DEX group.These results suggest that,chronic stress induced by DEX causes circadian rhythm disturbance in goats,particularly in the gastrointestinal tract,and also indicate the important role of melatonin on regulating epithelial physiology in vivo.3 Effect of melatonin on intestinal damage induced by pyocyanin in miceForty 8-week-old C57BL6/J male mice were housed in 12 h artificial illumination at room temperature(20℃).Mice were randomly divided into 4 groups(n=10):(a)control group(Con);(b)MEL-treated group(MEL);(c)PCN-treated group(PCN);(d)PCN&melatonin-treated group(PCN&MEL).PCN(600 μg/3days)was treated by gavage,and MEL(0.4 mg/mL)was added into drinking water.After 3 weeks of treatment,mice were slaughtered after 8 h fasting,and samples including blood,colonic mucosal epithelium and colonic digesta were collected for analysis.Compared to Con or MEL group,body weight of mice in PCN and PCN&MEL groups decreased significantly from the 2rd to 3th week administration(P<0.05),but MEL markedly alleviated the weight loss caused by PCN(P<0.05).PCN did not affect MEL secretion,however,plasma MEL in both MEL group and PCN&MEL group was significantly increased compared to Con(P<0.05).PCN can greatly activate inflammatory genes(IL-1,IL-8,iNOS,TNF-α,TLR4)expression,caspase 1/3 enzyme activity as well as NLRP3/Pro caspase-1 and NF-κB proteins expression in the colonic epithelium(P<0.05).However,in PCN&MEL group,MEL markedly suppressed inflammatory genes expression and these inflammasome related factors activation induced by PCN(P<0.05).Consistently,MEL effectively increased the antioxidative capacity by increasing GSH activity,and then to decrease ROS and MDA products in PCN&MEL treatment group compared to PCN group(P<0.05).Consistently,the up-regulation of apoptotic genes in PCN group was significantly inhibited by MEL co-treatment in PCN&MEL group(P<0.05).PCN treatment decreased colonic mucin 2(MUC2),tight junction proteins of claudin-1 and ZO-lexpression,while MEL treatment can effectively restored these structural proteins expression in PCN&MEL group(P<0.05).It’s very important to note that compared with the other three groups,PCN greatly inhibited GR/p-GR protein expression in the colonic epithelium,while in the PCN&MEL group,MEL inhibited the PCN induced down-regulation of GR/p-GR(P<0.05).The role of GR on the process of MEL alleviating the oxidative stress and epithelial injury induced by PCN is still unknown.4 Effect of melatonin on intestinal microecological imbalance induced by green pyocyanin in miceMicrobial genomic DNA was extracted from colonic digesta for 16S rRNA detection by Illumina Miseq.QIIME 1.9.1 was applied to analyze the microbial composition.The top 10 species with the highest abundance in each group at the phylum level,and the species with higher relative abundance and their proportion were observed.Compared to Con,the relative abundance of Proteobacteria and Vermina in PCN group was increased,but the relative abundance of Bacteroideteswas significantly decreased.In PCN&MEL group,MEL significantly inhibited the increase of relative abundance of Proteobacteriaand Vermina,and also prevented the decrease of relative abundance of Bacteroidetes induced by PCN.Among the top 50 genera in abundance were selected for further analysis.The results showed that in PCN&MEL group,MEL significantly inhibited the increase of Delftia,Pseudomonas,Burkholderiaceae and Akkermansia in Verruciformis abundance induced by PCN.Correlation analysis showed that the increased abundance of Pseudomonas aeruginosa was positively correlated with changes of body weight,histamine,LPS,inflammatory and apoptosis related genes expression in the colonic epithelium.Moreover,MEL alone treatment significantly increase the number of Odoribacter,Parabacteroides and Alistipesin Bacteroidetes;Lactobacillus and Erysipelatoclostridium in firmicutes in the colonic digesta.In summary,MEL significantly benefited the composition of microorganisms by increasing beneficial bacteria abundance and decreasing harmful bacteria abundance.Notably,MEL effectively inhibited the PCN-induced increase of Pseudomonas aeruginosa,and protected the intestinal injury from PCN by increasing the anti-oxidative capacity,inhibiting inflammasome activation as well as apoptosis related genes expression in the colonic epithelium.5 Role of glucocorticoid receptor on melatonin alleviating the apoptosis induced by PCN in goblet cellsThe aim of this experiment was to further investigate the cellular mechanism of PCN induced epithelial cells damage,and whether MEL achieves its anti-oxidative mainly through regulation of GR.The goblet cell line of LS174T was cultured in vitro.Cells were cultured and treated with DMSO(control,Con),PCN,MEL,PCN&MEL,PCN&MEL&RU486 and RU486 alone,respectively.The level of cell apoptosis,oxidative stress,inflammatory factors and other related indicators were determined and analyzed among different groups.Based on the data of cell viability,40 μM PCN,100 μM MEL and 3 nM RU486 were chosen as the optimal treated concentration in the following experiment.Compared with other groups,PCN significantly increased cellular oxidative response,indicating by high ROS level and caspase-1/3/9 enzyme activity(P<0.05),but low level of GSH activity,and thus led to higher cell apoptosis(P<0.05)but lower mucin secretion,however,in the PCN&MEL group MEL significantly decreased cellular oxidative stress,and inhibited cell apoptosis compared to PCN group(P<0.05).Two-way variance statistical analysis showed that there was a significant interaction between PCN and MEL.The electron microscopy showed that compared with the other groups,the vacuoles and swelling of mitochondria were induced by PCN,but these damages were inhibited by MEL in MEL&PCN group.Moreover,the expression of GR/p-GR in cells was significantly inhibited in PCN group(P<0.05),while MEL effectively alleviated the inhibitory effect of PCN on GR expression(P<0.05).RU486(3 nM)as a GR inhibitor,was added into the culture medium to interfere GR signal.The results showed that,after interfering GR signal by RU486,MEL’s alleviating effect on PCN-induced oxidative damages and apoptosis was significantly decreased compared with PCN&MEL group(P<0.05).Similarly,MEL’s inhibitory effect on PCN-induced the activation of NLRP3/Pro caspase-1,p-P65,p-IKB and inflammatory factors was also significantly decreased by RU486(P<0.05).In summary,in vivo and in vitro data showed that PCN induced severe oxidative damages and apoptosis in colonic goblet cells,which can be effectively alleviated by MEL,and MEL achieves its anti-oxidative and anti-apoptotic effects mainly through regulation of GR.This study will be helpful for understanding that the glucocorticoid receptor may be a target molecular for toxin attacking and the relationship between MEL and GR.